A novel AmBF₃-succinimide prosthetic group for facile ¹⁸F-labeling of biomolecules

Objectives Radiolabeled biomolecules like peptides and antibodies have widespread diagnostic and therapeutic applications. Due to its suitable half-life, ideal emission energies, and ease of production, fluorine-18 (¹⁸F) is often incorporated into the design of PET imaging agents. Different radioprosthetic groups like N-succinimidyl-4-[¹⁸F]fluorobenzoate have been used extensively for labeling biomolecules such as antibody fragments that are heat-sensitive and not compatible with organic solvents. However, the preparation of N-succinimidyl-4-[¹⁸F]fluorobenzoate require multiple synthesis steps. Leveraging the one step ¹⁸F-¹⁹F isotope exchange reaction, we synthesized succinimido 4-(N-trifluoroborylmethyl-N,N-dimethylammonio)methylbenzoate (AmBF₃-succinimide) and evaluated its ability to radiofluorinate selected biomolecules.

Methods The AmBF₃-succinimide was synthesized in four steps: coupling of tert-butyl 4-(bromomethyl)benzoate with dimethylamine, formation of quaternary ammonium iodide salt with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,4-dioxaborolane, deprotection and simultaneous conversion to trifluoroborate using HCl/KHF₂, and the final formation of succinimide with diisopropylcarbodiimide and N-hydroxysuccinimide. For radiolabeling, 100 nmol of ¹⁹F-AmBF₃-succinimide was re-suspended in a mixture of DMF and pyridazine buffer (1 M, pH 2.0). ¹⁸F-fluoride (~1 Ci ) was eluted off from a QMA cartridge with saline to the precursor solution. The solution was incubated at 85°C for 20 min, and purified by solid phase extraction using a C-18 sep-pak cartridge. To test its suitability for labeling biomolecules, ¹⁸F-AmBF₃-succinimide (~ 5 mCi) was added to a solution of Cetuximab (100 µg) in PBS (pH 8.9, 100 µL), and the reaction mixture was incubated at 37 °C for 30 min and purified by PD-10 column.

Results The overall synthesis yield for the non-radioactive ¹⁹F-AmBF₃-succinimide was 15% for four steps. ¹⁸F-AmBF₃-succinimide was obtained directly in aqueous solution via ¹⁸F-¹⁹F isotope exchange with 9-16 % decay-corrected radiochemical yield. ¹⁸F-AmBF₃-succinimide was successfully conjugated to Cetuximab in mild conditions. After PD-10 column purification, ¹⁸F-AmBF₃-Cetuximab was obtained in 20-22 % decay-corrected radiochemical yield from ¹⁸F-AmBF₃-succinimide with >97 % radiochemical purity and 44-48 GBq/µmol specific activity.

Conclusions ¹⁸F-AmBF₃-succinimide was successfully synthesized and characterized as a radioprosthetic group. For proof of concept, we selected Cetuximab as a surrogate biomolecule for conjugation. Our preliminary data suggests that ¹⁸F-AmBF₃-succinimide may be an attractive option for the radiofluorination of biomolecules for PET imaging.

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